Melting Curve Analysis after T Allele Enrichment (MelcaTle) as a Highly Sensitive and Reliable Method for Detecting the JAK2V617F Mutation

Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs). However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle). MelcaTle comprises three steps: 1) two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2) selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA) probe, and 3) a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.     

A tetra-primer polymerase chain reaction approach for the detection of JAK2 V617F mutation

Recently, an acquired somatic point mutation (p.V617F) in a highly conserved residue of the pseudokinase domain of the JAK2 tyrosine kinase was shown to be associated with myeloproliferative disorders. Because of the clinical importance of this mutation in diagnosing myeloproliferative disorders and its relevance for disease progression, we have developed a tetra-primer polymerase chain reaction (PCR) assay to detect JAK2 p.V617F. Titration studies showed that the assay could reliably detect one copy of the mutant allele in a mix of 50 wild-type alleles suggesting that the lower detection limit of this assay is estimated to be 2%. This study demonstrates that genotyping and quantifying of the JAK2 V617F mutation can be performed by tetra-primer PCR using both freshly isolated and formalin-fixed tissues. Our tetra-primer PCR assay is sensitive, low-cost, and easy to use method for the detection of JAK2 p.V617F, which could be used even in low-tech laboratories.     

Identification of oncostatin M as a JAK2 V617F-dependent amplifier of cytokine production and bone marrow remodeling in myeloproliferative neoplasms

The JAK2 mutation V617F is detectable in a majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Enforced expression of JAK2 V617F in mice induces myeloproliferation and bone marrow (BM) fibrosis, suggesting a causal role for the JAK2 mutant in the pathogenesis of MPNs. However, little is known about mechanisms and effector molecules contributing to JAK2 V617F-induced myeloproliferation and fibrosis. We show that JAK2 V617F promotes expression of oncostatin M (OSM) in neoplastic myeloid cells. Correspondingly, OSM mRNA levels were increased in the BM of patients with MPNs (median 287% of ABL, range 22-1450%) compared to control patients (median 59% of ABL, range 12-264%; P < 0.0001). OSM secreted by JAK2 V617F+ cells stimulated growth of fibroblasts and microvascular endothelial cells and induced the production of angiogenic and profibrogenic cytokines (HGF, VEGF, and SDF-1) in BM fibroblasts. All effects of MPN cell-derived OSM were blocked by a neutralizing anti-OSM antibody, whereas the production of OSM in MPN cells was suppressed by a pharmacologic JAK2 inhibitor or RNAi-mediated knockdown of JAK2. In summary, JAK2 V617F-mediated up-regulation of OSM may contribute to fibrosis, neoangiogenesis, and the cytokine storm observed in MPNs, suggesting that OSM might serve as a novel therapeutic target molecule in these neoplasms.     

Molecular drug targets in myeloproliferative neoplasms: mutant ABL1, JAK2, MPL, KIT, PDGFRA, PDGFRB and FGFR1

Therapeutically validated oncoproteins in myeloproliferative neoplasms (MPN) include BCR-ABL1 and rearranged PDGFR proteins. The latter are products of intra- ( e.g. FIP1L1-PDGFRA) or inter-chromosomal ( e.g. ETV6-PDGFRB ) gene fusions. BCR-ABL1 is associated with chronic myelogenous leukaemia (CML) and mutant PDGFR with an MPN phenotype characterized by eosinophilia and in addition, in case of FIP1L1-PDGFRA, bone marrow mastocytosis. These genotype-phenotype associations have been effectively exploited in the development of highly accurate diagnostic assays and molecular targeted therapy. It is hoped that the same will happen in other MPN with specific genetic alterations: polycythemia vera ( JAK2 V617F and other JAK2 mutations), essential thrombocythemia ( JAK2 V617F and MPL5 15 mutations), primary myelofibrosis ( JAK2 V617F and MPL515 mutations), systemic mastocytosis ( KIT D816V and other KIT mutations) and stem cell leukaemia/lymphoma ( ZNF198-FGFR1 and other FGFR1 fusion genes). The current review discusses the above-listed mutant molecules in the context of their value as drug targets.     

A highly sensitive quantitative real-time PCR assay for determination of mutant JAK2 exon 12 allele burden

Mutations in the Janus kinase 2 (JAK2) gene have become an important identifier for the Philadelphia-chromosome negative chronic myeloproliferative neoplasms. In contrast to the JAK2V617F mutation, the large number of JAK2 exon 12 mutations has challenged the development of quantitative assays. We present a highly sensitive real-time quantitative PCR assay for determination of the mutant allele burden of JAK2 exon 12 mutations. In combination with high resolution melting analysis and sequencing the assay identified six patients carrying previously described JAK2 exon 12 mutations and one novel mutation. Two patients were homozygous with a high mutant allele burden, whereas one of the heterozygous patients had a very low mutant allele burden. The allele burden in the peripheral blood resembled that of the bone marrow, except for the patient with low allele burden. Myeloid and lymphoid cell populations were isolated by cell sorting and quantitative PCR revealed similar mutant allele burdens in CD16+ granulocytes and peripheral blood. The mutations were also detected in B-lymphocytes in half of the patients at a low allele burden. In conclusion, our highly sensitive assay provides an important tool for quantitative monitoring of the mutant allele burden and accordingly also for determining the impact of treatment with interferon-α-2, shown to induce molecular remission in JAK2V617F-positive patients, which may be a future treatment option for JAK2 exon 12-positive patients as well.     

Impact of JAK2 V617F Mutation on Hemogram Variation in Patients with Non-Reactive Elevated Platelet Counts

Background

Non-reactive platelet counts elevation occurs mainly in myeloproliferative disorders (MPDs), which have been reported to be closely associated with JAK2 V617F mutation. Complete blood cell count (CBC) is essential in diagnosis of MPDs, however, the impact of JAK2 V617F mutation on the patients’ hemogram variation remains not clear.

Methods

JAK2 V617F mutation was detected by allele specific real-time quantitative fluorescence PCR (AS-qPCR).

Results

Of the 402 non-reactive platelet elevating patients, JAK2 V617F mutation was detected in 222 (55.2%) patients. RBC counts, WBC counts, platelet-large contrast ratio (P-LCR), platelet distribution width (PDW) and mean platelet volume (MPV) were much higher in JAK2 V617F mutated patients, except platelet counts. In addition, when the patients were classified into subgroups by blood cell counts, it was found that JAK2 V617F mutation rate increased progressively with the increase of RBC counts and WBC counts, other than platelet counts. Furthermore, trilineage hyperplasia group showed highest JAK2 V617F mutation rate (93.26%), followed by the bilineage hyperplasia groups. Lastly, JAK2 V617F mutant allele burden was found much higher in polycythemia vera (PV) patients [median(P₂₅–P₇₅): 45.02%(35.12%–54.22%)] than in essential thrombocythemia (ET) patients [median(P₂₅–P₇₅): 28.23%(17.77%–41.66%)], and that it increased with WBC counts (r = 0.393, p = 0.000) and RBC counts(r = 0.215, p = 0.001), other than platelet counts (r = −0.051, p = 0.452). Further analysis revealed that in ET patients, JAK2 V617F mutant allele burden correlated with WBC counts and platelet counts positively, other than RBC counts, while in PV patients, it correlated with WBC counts and RBC counts positively, but not platelet counts.

Conclusions

JAK2 V617F mutation occurs frequently in patients with non-reactive elevated platelet counts. The presence of JAK2 V617F mutation has great impact on hemogram variation, including RBC counts, WBC counts, platelet parameters and lineage hyperplasia, but not on platelet counts. Besides, JAK2 V617F mutant allele burden affects the blood cell proliferation pattern.     

Combination treatment for myeloproliferative neoplasms using JAK and pan‐class I PI3K inhibitors

Current JAK2 inhibitors used for myeloproliferative neoplasms (MPN) treatment are not specific enough to selectively suppress aberrant JAK2 signalling and preserve physiological JAK2 signalling. We tested whether combining a JAK2 inhibitor with a series of serine threonine kinase inhibitors, targeting nine signalling pathways and already used in clinical trials, synergized in inhibiting growth of haematopoietic cells expressing mutant and wild‐type forms of JAK2 (V617F) or thrombopoietin receptor (W515L). Out of 15 kinase inhibitors, the ZSTK474 phosphatydylinositol‐3′‐kinase (PI3K) inhibitor molecule showed strong synergic inhibition by Chou and Talalay analysis with JAK2 and JAK2/JAK1 inhibitors. Other pan‐class I, but not gamma or delta specific PI3K inhibitors, also synergized with JAK2 inhibitors. Synergy was not observed in Bcr‐Abl transformed cells. The best JAK2/JAK1 and PI3K inhibitor combination pair (ruxolitinib and GDC0941) reduces spleen weight in nude mice inoculated with Ba/F3 cells expressing TpoR and JAK2 V617F. It also exerted strong inhibitory effects on erythropoietin‐independent erythroid colonies from MPN patients and JAK2 V617F knock‐in mice, where at certain doses, a preferential inhibition of JAK2 V617F mutated progenitors was detected. Our data support the use of a combination of JAK2 and pan‐class I PI3K inhibitors in the treatment of MPNs.     

Evaluation of clinical and laboratory findings with JAK2 V617F mutation as an independent variable in essential thrombocytosis

Essential thrombocythemia (ET) is an entity of classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), characterized by thrombocytosis with megakaryocytic hyperplasia and thrombocytes are increased with abnormal functions. Discovery of the protein tyrosine kinase JAK2 V617F allele contributed to better understanding of the pathogenetic mechanisms of MPNs. Acquired single point mutation in the JAK2 V617F was determined approximately 50–60 % of patients with ET. In this study we aimed to investigate the relationship between JAK2 V617F gene mutation, hematologic, biochemical markers and the complications in the ET patients. A total of 268 patients diagnosed with ET and 219 of those studied for JAK2 gene mutation were followed at the hematology clinics of three major hospitals between 2008 and 2013 were screened retrospectively. Laboratory, clinical and hematologic parameters were compared for JAK2 V617F positive and JAK2 V617F negative patients with ET. 102 (46 %) patients were positive with the JAK2 V617F mutation. The complications were observed in 61 (28 %) patients and 38 (62 %) of them had JAK2 V617F mutation. The levels of white blood cells, neutrophil, basophil, red blood cells, hemoglobin, hematocrit, mean platelet volume, thrombocytes, eosinophil; urea, creatinine were significantly different in patients with the JAK2 V617F mutation (P < 0.05). Presence of the JAK2 V617F mutation supports the diagnosis of ET. It would be useful to investigate the JAK2 V617F mutation and the hematologic and biochemical markers at diagnosis with respect to consider the risk of developing complications and to take the precautions against these complications.     

Fullerene derivative prevents cellular transformation induced by JAK2 V617F mutant through inhibiting c-Jun N-terminal kinase pathway

The constitutively activated mutation (V617F) of tyrosine kinase Janus kinase 2 (JAK2) is found in the majority of patients with myeloproliferative neoplasms (MPNs). The development of a novel chemical compound to suppress JAK2 V617F mutant-induced onset of MPNs and clarification of the signaling cascade downstream of JAK2 V617F mutant will provide clues to treat MPNs. Here we found that a water-soluble pyrrolidinium fullerene derivative, C(60)-bis (N, N-dimethylpyrrolidinium iodide), markedly induced apoptosis of JAK2 V617F mutant-induced transformed cells through a novel mechanism, inhibiting c-Jun N-terminal kinase (JNK) activation pathway but not generation of reactive oxygen species (ROS). Pyrrolidinium fullerene derivative significantly reduced the protein expression level of apoptosis signal-regulating kinase 1 (ASK1), one of the mitogen-activated protein kinase kinase kinases (MAPKKK), resulting in the inhibition of upstream molecules of JNK, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7). Strikingly, the knockdown of ASK1 enhanced the sensitivity to pyrrolidinium fullerene derivative-induced apoptosis, and the treatment with a JNK inhibitor, SP600125, also induced apoptosis of the transformed cells by JAK2 V617F mutant. Furthermore, administration of both SP600125 and pyrrolidinium fullerene derivative markedly inhibited JAK2 V617F mutant-induced tumorigenesis in nude mice. Taking these findings together, JAK2 V617F mutant-induced JNK signaling pathway is an attractive target for MPN therapy, and pyrrolidinium fullerene derivative is now considered a candidate potent drug for MPNs.     

The Polymorphisms in LNK Gene Correlated to the Clinical Type of Myeloproliferative Neoplasms

ObjectiveLNK is an adapter protein negatively regulating the JAK/STAT cell signaling pathway. In this study, we observed the correlation between variation in LNK gene and the clinical type of myeloproliferative neoplasms (MPN).MethodsA total of 285 MPN cases were recruited, including essential thrombocythemia (ET) 154 cases, polycythemia vera (PV) 76 cases, primary myelofibrosis (PMF) 19 cases, and chronic myeloid leukemia (CML) 36 cases. Ninety-three healthy individuals were used as normal controls. V617F mutation in JAK2 was identified by allele-specific PCR method, RT-PCR was used for the detection of BCR/ABL1 fusion gene, and mutations and variations in coding exons and their flanking sequences of LNK gene were examined by PCR-sequencing.ResultsMissense mutations of A300V, V402M, and R415H in LNK were found in 8 patients including ET (4 cases, all combined with JAK2-V617F mutation), PV (2 cases, one combined with JAK2-V617F mutation), PMF (one case, combined with JAK2-V617F mutation) and CML (one case, combined with BCR/ABL1 fusion gene). The genotype and allele frequencies of the three SNPs (rs3184504, rs111340708 and rs78894077) in LNK were significantly different between MPN patients and controls. For rs3184504 (T/C, in exon2), the T allele (p.262W) and TT genotype were frequently seen in ET, PV and PMF (P<0.01), and C allele (p.262R) and CC genotype were frequently seen in CML (P<0.01). For rs78894077 (T/C, in exon1), the T allele (p.242S) was frequently found in ET (P<0.05). For rs111340708 (TGGGGx5/TGGGGx4, in intron 5), the TGGGG x4 allele was infrequently found in ET, PMF and CML(P<0.01).ConclusionMutations in LNK could be found in some of MPN patients in the presence or absence of JAK2-V617F mutation. Several polymorphisms in LNK gene may affect the clinical type or the genetic predisposition of MPN.     

JAK2V617F 点突变与骨髓增殖性疾病的临床相关性研究

目的:研究JAK2V617F点突变与骨髓增殖性疾病(myeloproliferative disease,MPD)的临床相关性,为MPD的基因学诊断及靶向治疗提供理论依据。方法:应用等位基因特异性聚合酶链反应(AS-PCR)检测JAK2V617F点突变。结果:102例的MPD患者中包括慢性粒细胞白血病(CML)患者9例、真性红细胞增多症(PV)患者21例、原发性血小板增多症(ET)患者37例、特发性骨髓纤维化(IMF)患者16例和分类不明的骨髓增殖性疾病(uMPD)患者19例,JAK2V617F突变阳性率依次为11%、71.4%、51.4%、75.0%、78.9%。结论:JAK2V617F点突变有助于不同类型MPD的诊断,在MPD疾病的诊断中起重要作用。     

Co‐targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms

Aberrant JAK2 signalling plays a central role in myeloproliferative neoplasms (MPN). JAK2 inhibitors have proven to be clinically efficacious, however, they are not mutation‐specific and competent enough to suppress neoplastic clonal haematopoiesis. We hypothesized that, by simultaneously targeting multiple activated signalling pathways, MPN could be more effectively treated. To this end we investigated the efficacy of BEZ235, a dual PI3K/mTOR inhibitor, alone and in combination with the JAK1/JAK2 inhibitor ruxolitinib, in different preclinical models of MPN. Single‐agent BEZ235 inhibited the proliferation and induced cell cycle arrest and apoptosis of mouse and human JAK2V617F mutated cell lines at concentrations significantly lower than those required to inhibit the wild‐type counterpart, and preferentially prevented colony formation from JAK2V617F knock‐in mice and patients' progenitor cells compared with normal ones. Co‐treatment of BEZ235 and ruxolitinib produced significant synergism in all these in‐vitro models. Co‐treatment was also more effective than single drugs in reducing the extent of disease and prolonging survival of immunodeficient mice injected with JAK2V617F‐mutated Ba/F3‐EPOR cells and in reducing spleen size, decreasing reticulocyte count and improving spleen histopathology in conditional JAK2V617F knock‐in mice. In conclusion, combined inhibition of PI3K/mTOR and JAK2 signalling may represent a novel therapeutic strategy in MPN.     

Akt activation through the phosphorylation of erythropoietin receptor at tyrosine 479 is required for myeloproliferative disorder-associated JAK2 V617F mutant-induced cellular transformation

The disruption of Janus kinase 2 (JAK2) signaling regulation by its point mutation, V617F, is involved in various myeloproliferative disorders (MPDs). JAK2 V617F mutant induced constitutive activation of Akt when erythropoietin receptor (EpoR) was coexpressed; however, the physiological role of Akt activation in MPDs has not been elucidated. LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, inhibited Akt activation and induced apoptotic cell death in cells expressing JAK2 V617F mutant and EpoR. Previously, it has been shown that the phosphorylation at Y479 in EpoR is critical for the interaction with PI3K, an upstream molecule of Akt. Hence, EpoR mutant with a point mutation of Y479F, which fails to activate Akt, is useful for addressing the role of Akt activation in JAK2 V617F mutant-induced tumorigenesis. Interestingly, under the expression of EpoR Y479F mutant, JAK2 V617F mutant failed to exhibit potent anti-apoptotic activity. In addition, JAK2 V617F mutant-induced phosphorylation of CREB and GSK-3β was significantly decreased in cells expressing EpoR Y479F mutant, resulting in the downregulation of Bcl-XL and Mcl-1 expression. Furthermore, compared with when nude mice were inoculated with cells expressing JAK2 V617F mutant and EpoR, the lifespan of nude mice inoculated with cells expressing JAK2 V617F mutant and EpoR Y479F mutant was effectively prolonged. Taken together, it was clarified that PI3K-Akt activation through the phosphorylation of EpoR at Y479 is required for oncogenic signaling of JAK2 V617F mutant and that targeted disruption of this pathway has therapeutic utility.     

Epigenetic dysregulation of secreted frizzled-related proteins in myeloproliferative neoplasms complements the JAK2V617F-mutation

ABSTRACT: BACKGROUND: Secreted frizzled-related proteins (SFRPs) are antagonists of the Wnt signaling pathway, which plays a central role in stem cell maintenance and differentiation of stem cells and hematopoietic progenitors. Epigenetic downregulation of SFRPs by promoter hypermethylation has been described to be involved in the pathogenesis of hematopoietic malignancies. There is an association between aberrant Wnt signaling and the established cancer stem cell concept. In contrast to BCR-ABL1-positive chronic myeloid leukemia, BCR-ABL1-negative myeloproliferative neoplasms (Ph-MPN) are characterized by the frequent occurrence of an autoactivating mutation in the JAK2 tyrosine kinase (JAK2V617F) or other mutations in the JAK-STAT pathway. However, pathogenetic mechanisms of JAK2 mutated or unmutated Ph-MPN remain not completely understood.We determined the promoter methylation status of SFRP-1, -2, -4, and [MINUS SIGN]5 in 57 MPN patient samples by methylation-specific polymerase chain reaction (PCR) (MSP). JAK2V617F was assessed by allele-specific PCR. RESULTS: Aberrant methylation among primary MPN samples was 4% for SFRP-1, 25% for SFRP-2, 2% for SFRP-4, and 0% for SFRP-5. Hypermethylation of SFRP-2, which was the most frequently hypermethylated gene in our study, could not be correlated to any specific MPN subtype. However, we detected a significant correlation between SFRP-2 methylation and presence of a JAK2V617F mutation (P = 0.008). None of the 10 CML samples showed any SFRP-methylation. CONCLUSIONS: Our data indicate that epigenetic dysregulation of the Wnt signaling pathway is a common event in MPN with aberrant methylation of at least one SFRP being detected in 25% of the primary patient samples and in 30% if only accounting for Ph-MPN. A significant correlation between SFRP-2 methylation and presence of JAK2V617F in our data support the hypothesis that epigenetic dysregulation may be a complementary mechanism to genetic aberrations. Aberrant methylation of crucial stem cell maintenance genes seems to contribute to disease pathogenesis in Ph-MPN.     

Improved Diagnosis of the Transition to JAK2 V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms

Most cases of BCR-ABL1-negative myeloproliferative neoplasms (MPNs), essential thrombocythemia, polycythemia vera and primary myelofibrosis are associated with JAK2 ^V617F mutations. The outcomes of these cases are critically influenced by the transition from JAK2 ^V617F heterozygosity to homozygosity. Therefore, a technique providing an unbiased assessment of the critical allele burden, 50% JAK2 ^V617F, is highly desirable. In this study, we present an approach to assess the JAK2 ^V617F burden from genomic DNA (gDNA) and complementary DNA (cDNA) using one-plus-one template references for allele-specific quantitative-real-time-PCR (qPCR). Plasmidic gDNA and cDNA constructs encompassing one PCR template for JAK2 ^V617F spaced from one template for JAK2^{Wild Type} were constructed by multiple fusion PCR amplifications. Repeated assessments of the 50% JAK2^V617F burden within the dynamic range of serial dilutions of gDNA and cDNA constructs resulted in 52.53±4.2% and 51.46±4.21%, respectively. The mutation-positive cutoff was estimated to be 3.65% (mean +2 standard deviation) using 20 samples from a healthy population. This qPCR approach was compared with the qualitative ARMS-PCR technique and with two standard methods based on qPCR, and highly significant correlations were obtained in all cases. qPCR assays were performed on paired gDNA/cDNA samples from 20 MPN patients, and the JAK2 ^V617F expression showed a significant correlation with the allele burden. Our data demonstrate that the qPCR method using one-plus-one template references provides an improved assessment of the clinically relevant transition of JAK2 ^V617F from heterozygosity to homozygosity.